INTRODUCTION: We have shown that mast cell degranulation is dependent on a rise in intracellular calcium (calcium signal)in concert with activation of protein kinase (PK) C and phospholipase(PL)D. Least is known about the activation and role of PLD in degranulation and we have focussed our attention on this enzyme in recent years. We and others have shown that PLD is activated in isolated mast cells and a variety of cultured mast cell lines by antigen and other stimulants. The correlations between PLD activity and mast cell degranulation under a variety of experimental conditions as well the fact that primary alcohols, which divert production of phosphatidic acid to the corresponding phosphatidyl alcohol, suppress degranulation suggest that PLD has a critical role in degranulation. We have shown, by transient expression of either of the two known mammalian isoforms of PLD in the RBL-2H3 mast cell line, that PLD1 and PLD2 associate with granule membranes and the plasma membrane, respectively (Choi et al., J. Immunol. 278:12039, 2002). Also, both isoforms are activated upon antigen stimulation and regulate different phases of degranulation in mast cells, granule-associated PLD1 in the translocation of granules to the plasma membrane and plasma membrane-associated PLD2 in the fusion of granules with the plasma membrane. However, the mechanisms by which PLD is activated and its downstream targets remain largely undefined. The handicap in research on PLD is the lack of suitable antibodies with high affinity or selectivity for the PLDs, gene-deficient mice, and pharmacologic inhibitors with the exception of primary alcohols as noted above. For this reason, we have resorted to overexpression of wild type, mutant, and tagged PLDs as well as siRNAs directed against the PLDs. OBJECTIVES: We are pusuing two lines of investigation: the first is to elucidate the mechanisms of activation of PLD and the second to identify downstream events that are regulated by PLD. PLD2 is of particular interest because it location at the plasma membrane makes this isoform accessible to Fc{epsilon}RI-associated Src kinases and other signaling molecules that are recruited during the activation process. It should be noted that, in the allergic condition, mast cells are activated by multivalent binding of antigen to immunoglobulin E (IgE) that is bound to its multimeric receptor, Fc{epsilon}RI. The ensuing aggregation of these receptors results in the rapid phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc{epsilon}RI subunits by the Src kinase, Lyn, and as a consequence the recruitment and activation of a second protein tyrosine kinase, Syk. Syk, in turn, is responsible for the phosphorylation/activation/recruitment of a variety of downstream adaptor and effector molecules and thus regulates downstream signals for the release and generation of inflammatory mediators. Therefore, we have investigated the potential interaction of PLD2 with all of these molecules by a variety of techniques. Further, with respect to downstream events, we have examined the role of PLD in the activation of PKC because the PLD product, phosphatidic acid, is rapidly converted to diglycerides which could potentially activate diglyceride-dependent isoforms of PKC. ACTIVATION OF PLD2 BY SRC KINASES IS REQUIRED FOR DEGRANULATION: We found that PLD2 but not PLD1 is phosphorylated by the Src kinases, Fyn and Fgr, and this phosphorylation regulates PLD2 activation and degranulation. For example, only hemagglutinin (HA)-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by Src kinase inhibitors or by siRNAs directed against Fyn and Fgr and was enhanced by overexpresssion of Fyn and Fgr but not by other Src kinases. Mutation of PLD2 at phosphorylated tyrosines (11, 14, 165, or 44470) partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation as well as degranulation. Kinetic studies revealed that PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by co-expression of PLD2 and the Src kinases. The findings indicate a novel mechanism for activation of PLD2 in a physiological setting and point to a role for Fgr in Fc{epsilon}RI-mediated signaling (Ref.1). PLD2 INTERACTS WITH SYK AND REGULATES SYK ACTIVITY AND DOWNSTREAM EVENTS: We have also found that PLD2 associates with and promotes activation of Syk which, as noted above is a key enzyme in mast cell activation. Antigen stimulation resulted in increased co-localization of PLD2 with Syk and and the adaptor protein LAT on the plasma membrane, probably in association with lipid rafts, as indicated by co-immunoprecipitation studies, confocal microscopy, and other techniques. This association was dependent on tyrosine phosphorylation of Syk by Lyn, but not on PLD2 activity. Both in vivo and in vitro, the interaction of PLD2 with Syk induced additional phosphorylation and and was necessary for the activation of Syk. Furthermore, overexpression of PLD2 or catalytically inactive PLD2K758R enhanced antigen-induced phosphorylations of Syk and its downstream targets, the adaptor proteins LAT and SLP-76, while expression of a PLD2 siRNA blocked these phosphorylations. Apparently, the interaction of PLD2 with Syk is an early critical event in the activation of mast cells (manuscript submitted). PLD IS ESSENTIAL FOR ACTIVATION OF DIGLYCERIDE-DEPENDENT ISOFORMS OF PKC AND DEGRANULATION: We tested the hypothesis that the PLD product, phosphatidic acid and diacylglycerides generated therefrom, activate diglyceride-dependent forms of PKC. Two rodent mast cell lines were stimulated with antigen and a pharmacologic stimulant, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol by addition of 1-butanol suppressed the translocation of diacylglyceride-dependent, but not diacylglyceride-independent isoforms of PKC, to the membrane as well as degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had minimal effect on PKC translocation and degranulation and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with the diacylglycerol analogue, 12-O-tetradecanoylphorbol-13-acetate (phorbol ester). Also, in cells transfected with siRNAs directed against PLD1 and PLD2, activation of PLD, generation of phosphatidic acid and diacylglycerides, translocation of PKC, and degranulation were all suppressed. Direct stimulation of PKC by phorbol ester, which by itself does not stimulate degranulation, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the siRNAs. However, degranulation was not restored when cells were co-stimulated with antigen and phorbol ester. We conclude that production of phosphatidic acid by PLD is the primary source of diacylglycerides in mast cells and is essential for sustained activation of PKC and degranulation. However, additional PLD-dependent processes appear to be necessary for antigen-mediated degranulation (Ref. 2). One such process could be the activation of Syk as described above. Collectively, the studies thus far point to pluralistic roles for PLD in degranulation, one attributed to its ability to interact directly with tyrosine kinases and others to its ability to generate biologically active lipids that regulate activation of PKC and potentially other regulatory molecules